Effects of Zinc Finger Protein A20 on Lipopolysaccharide (LPS)-Induced Pulmonary Inflammation/Anti-Inflammatory Mediators in an Acute Lung Injury/Acute Respiratory Distress Syndrome Rat Model

Abstract

BACKGROUND The aim of this study was to investigate the effects of zinc finger protein A20 on lipopolysaccharide (LPS)-induced pulmonary inflammation/anti-inflammatory mediators in an acute lung injury/acute respiratory distress syndrome (ALI/ARDS) rat model. MATERIAL AND METHODS Forty-eight ALI/ARDS rats were selected and assigned into normal saline (NS) (injected with NS), LPS (injected with LPS), LPS-C1 (injected with pEGFP-C1, NS and LPS), and A20 groups (injected with pEGFP-C1-A20, NS, and LPS). The wet/dry (W/D) ratio of rat lung tissues and total protein concentration and the number of neutrophils in bronchoalveolar lavage fluid (BALF) were detected. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR were applied to detect the protein and mRNA expressions of A20, IL-10, and TNF-α, respectively. Western blotting was employed to detect the protein expressions of A20, nuclear factor-kappa B (NF-κB) p65 and NF-κB p-P65 in rat lung tissues. RESULTS Compared with the NS group, the W/D ratio of rat lung tissues and total protein concentration and the number of neutrophils in BALF in the other 3 groups increased significantly. The protein and mRNA expressions of A20, IL-10, and TNF-α were significantly higher in the LPS group than in the NS group. The protein and mRNA expressions of A20 and IL-10 were significantly up-regulated and the expression of TNF-α, NF-κB p65, and NF-κB p-P65 was significantly down-regulated in rats injected with A20 compared to those in the LPS group. CONCLUSIONS The study provided evidence that zinc finger protein A20 can alleviate pulmonary inflammation by inhibiting TNF-α, NF-κB p65, and NF-κB p-P65 expressions and promoting IL-10 expression.

Figure 1

Double-enzyme digestion and identification of vector pEGFP-C1-A20. (A) Double-enzyme digestion of A20 pUC57 plasmid; M, Marker; I, double-enzyme digestion of Xba I/BamH I; (B) Double-enzyme digestion of A20 pEGFP-C1 plasmid; M – Marker; I – double-enzyme digestion of BamH1/Bgl II.

Figure 2

Comparisons of W/D ratio in rat lung tissues and total protein concentration and the number of neutrophils in BLAF among 4 groups. (A) W/D ratio in rat lung tissues; (B) total protein concentration in BLAF (μg/mL); (C) the number of neutrophils in BLAF; BLAF – bronchoalveolar lavage fluid; NS – normal saline; LPS – lipopolysaccharide; C1 – pEGFP-C1; * P<0.05 compared with the NS group; ^# P<0.05 compared with the LPS group; Experimental data are presented as mean ± standard deviation (SD); n=12.

Figure 3

HE staining images of rat left lung tissues 24 h after injection with NS or LPS among 4 groups (400× magnification). NS – normal saline; LPS – lipopolysaccharide; C1 – pEGFP-C1; (A) the NS group; (B) the LPS group; (C) the LPS-C1 group; (D) the A20 group; n=12.

Figure 4

Comparisons of zinc finger protein A20 expression in rat lung tissues 24 h after injection among 4 groups. NS – normal saline; LPS – lipopolysaccharide; C1 – pEGFP-C1; * P<0.05 compared with the NS group; ^# P<0.05 compared with the LPS group; n=12.

Figure 5

Comparisons of IL-10 (A) and TNF-α (B) expressions in rat lung tissues 24 h after injection with NS or LPS among 4 groups. NS – normal saline; LPS – lipopolysaccharide; C1 – pEGFP-C1; * P<0.05 compared with the NS group; ^# P<0.05 compared with the LPS group; n=12.

Figure 6

(A–C) Comparisons of relative mRNA expressions of A20, TNF-α, and IL-10 in rat lung tissues among 4 groups. NS – normal saline; LPS – lipopolysaccharide; C1 – pEGFP-C1; ^# P<0.05 compared with the LPS group, n=12.

Figure 7

Comparisons of protein expressions of A20, NF-κB p65, and NF-κB p-P65 in rat lung tissues among 4 groups. NS – normal saline; LPS – lipopolysaccharide; C1 – pEGFP-C1.